This revised application is a request for renewal of R01 DA020686. A major accomplishment of the previous funding period was identifying a key for ?5-containing nicotinic acetylcholine receptors (?5* nAChRs) in the habenulo-interpeduncular tract in regulating nicotine intake. We found that nicotine-induced activation of ?5* nAChRs in the habenulo-interpeduncular tract triggered a negative motivational signal that served to reduce further nicotine consumption. Genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the ?5, ?3 and 4 nAChR subunits respectively, increases vulnerability to tobacco addiction and smoking- associated diseases including lung cancer. Here, we will fully define the role for ?5*, ?3* and 4* nAChRs in the MHb-IPN tract regulating nicotine reinforcement. Under Specific Aim I, we will assess nicotine self-administration behavior in knock-in mice in which the ?5 nAChR subunit gene has been genetically modified to express a major risk allele for tobacco dependence in humans. Second, we will use an elegant combination of Double-floxed Inverted (DiO) adeno- associated viruses in Cre-expressing transgenic mice or in mice treated with Cre-expression virus to selectively re-express ?5 nAChR subunits in discrete neuronal populations and pathways in the brains of ?5 subunit knockout mice. We will then assess the impact on nicotine self-administration behavior in these rescued mice. Under Specific Aim II we will investigate the role for ?3* nAChRs in the habenulo-interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that ?3 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. Under Specific Aim III we will investigate the role for 4* nAChRs in the habenulo- interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that 4 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will similarly confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. These studies promise to yield significantly new advances in our understanding of nicotine dependence and tobacco addiction.